摘要
背景:由于少突胶质细胞主要来自少突前体细胞,实验通过提取少突胶质前体细胞获取少突胶质细胞的过程中,选择合适的培养基和细胞接种密度对生长及形态学观察具有重要影响。目的:对比优化少突胶质细胞的培养条件。方法:取48 h内的SD新生鼠大脑皮质前体少突胶质细胞。分别用DMEM/高糖、DMEM/F12培养基培养少突胶质前体细胞,每组接种密度分别2×104/cm2,4×104/cm2,8×104/cm2,16×104/cm2,32×104/cm2,64×104/cm2;分别于少突胶质前体细胞贴壁72 h后,进行诱导分化,分化7 d后的少突胶质前体细胞在光镜下观察细胞形态变化并通过免疫荧光技术对细胞进行鉴定。结果与结论:DMEM/高糖、DMEM/F12培养基以2×104/cm2,4×104/cm2,8×104/cm2接种密度时,可辨识完整少突胶质前体细胞形态。诱导分化7 d后,免疫荧光鉴定提示3组少突胶质前体细胞均可呈现髓鞘碱性蛋白阳性,F12组中2×104/cm2,4×104/cm2,8×104/cm2接种密度条件下细胞均数分别为16.40±3.30,49.95±2.33,76.95±4.86,高于相应条件下的高糖组12.65±2.53,32.10±1.17,54.05±1.56(P<0.05)。结果表明,DMEM/F12较DMEM/高糖培养基适于少突胶质细胞培养,在一定范围内,少突胶质前体细胞分化为少突胶质细胞随接种密度成梯度增多,接种密度在4×104/cm2-8×104/cm2范围对观察细胞形态观察较为适合。
BACKGROUND：Oligodendrocytes are mostly differentiated from oligodendrocyte precursor cel s. A suitable medium and cel seeding density have a significant impact on the process of the isolation of oligodendrocyte precursor cel s to obtain oligodendrocytes. OBJECTIVE：To explore the optimization of oligodendrocyte culture conditions. METHODS：Oligodendrocyte precursor cel s isolated from the newborn rats 48 hours after birth were cultured in DMEM/high glucose medium or DMEM/F12 medium using seeding densities of 2×10^4 cel s/cm^2, 4×10^4 cel s/cm^2, 8×10^4 cel s/cm^2, 16×104 cel s/cm^2, 32×10^4 cel s/cm^2, and 64×10^4 cel s/cm^2, respectively. Oligodendrocyte precursor cel s were induced to differentiate into oligodendrocytes at 72 hours after cel adhesion. Morphology of differentiated oligodendrocyte precursor cel s were observed under a light microscope, and the differentiation results were identified by immunofluorescence staining after 7-day induced differentiation. RESULTS AND CONCLUSION：Morphology of oligodendrocyte precursor cel s were recognized when cultured in DMEM/high glucose medium or DMEM/F12 medium using seeding densities of 2×10^4 cel s/cm^2, 4×10^4 cel s/cm^2, and 8×10^4 cel s/cm^2, respectively. Immunofluorescence staining showed that myelin basic protein-positive cel s were found after 7-day induced differentiation, and the positive cel number were 16.40±3.30, 49.95±2.33, and 76.95±4.86 in DMEM/F12 medium, and 12.65±2.53, 32.10±1.17, and 54.05±1.56 in DMEM/high glucose medium （P〈0.05）. These findings indicate that DMEM/F12 medium is more suitable for culturing oligodendrocyte precursor cel s compared with DMEM/high glucose medium to some extent. The number of differentiated oligodendrocytes was gradual y increased with the enhanced seeding density of oligodendrocyte precursor cells, and the seeding densities from 4×10^4 to 8×10^4 cells/cm^2 were appropriate for the observation of cell morphology.
出处
《中国组织工程研究》
CAS
北大核心
2016年第29期4328-4333,共6页
Chinese Journal of Tissue Engineering Research
基金
国家自然科学基金(11102235)
天津市科技支撑项目(14ZCZDGX00500)
天津市卫生局课题(2013KZ134
2014KZ135)
武警后勤学院附属医院种子基金(FYM201417
FYM201432
FYM201542
WHJ2015018)
武警后勤学院博士启动基金(WHB201417
WHB201514)
武警后勤学院中心实验室开放基金(2015ZXKF01)~~
